Food waste reduction can be achieved by applying protective cultures to avoid spoilage of fermented food products. In this study, we present an approach to screen large numbers of strains for potential use as protective cultures in food. A phenotypic screening of 504 Lactobacillus strains for 27 food‐relevant growth conditions revealed variations and physiological limits for the genus. Previously, the strains were tested for their antibacterial activity in a high‐throughput assay. Here, the activity of 22 positive strains from that screening was assessed in more detail, mainly against Listeria, Enterococcus, Rhodotorula, and Candida species. The proteinaceous nature of the inhibiting substances was confirmed by protease digestion. Twenty‐two antibacterial and 42 antifungal strains were detected. In a co‐culture competition‐assay, 1–2 cfu/ml of Lactobacillus plantarum RI‐162 were able to inhibit the outgrowth of Rhodotorula mucilaginosa LME and reduce the cell number below the detection limit of 50 cfu/ml within 48 hr.